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dc.contributor.authorIbarra, Catherine Aen_US
dc.date.accessioned2009-10-06T21:18:22Z
dc.date.available2009-10-06T21:18:22Z
dc.date.issued2002en_US
dc.identifier.otherb49333276en_US
dc.identifier.other51962690en_US
dc.identifier.otherThesis 51803en_US
dc.identifier.urihttp://hdl.handle.net/1773/8186
dc.descriptionThesis (Ph. D.)--University of Washington, 2002en_US
dc.description.abstractAs the most abundant isoform of GST in the liver, the GST A1-1 is the major catalyst of detoxification. This isoform is of particular interest because it exhibits an unusually low pKa (∼8.1--8.3) of active site Tyr-9 and a ligand-dependent dynamics of the C-terminus. This dynamics is catalytically important, as product release is rate-limiting for a number of substrates.A major emphasis of this dissertation is the elucidation of the structural and functional significance of the anomalous pKa of Tyr-9 in rat GST A1-1. The edge-to-face electrostatic interaction between Phe-10 and Tyr-9 contributes to the ionization properties of the tyrosyl hydroxyl group in the apo-enzyme, consistent with crystal structure observations. Additionally, Phe-10 plays a vital role in ligand binding, as the two steps of the binding reaction are significantly reduced upon removal of aromatic-aromatic interactions. The results presented herein demonstrate the importance of Phe-10 in orchestrating the dynamics of the C-terminus.Previous data suggest that Tyr-9 ionization is closely coupled to the C-terminal dynamics of GST Al-1. To directly probe this possibility, engineered W21F:F222W and Y9F:W21F:F222W mutants, which contain single Trp at the C-terminus, were used as fluorescent reporters of C-terminal dynamics. Only the W21F:F222W mutant exhibited pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation. Moreover, the pH-dependent equilibrium binding and stopped-flow studies with product conjugate resulted in decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates at higher pHs. The recovered pKas for the change in microscopic rate constants reflect the pKa of Tyr-9 in the apo-enzyme. In contrast, these pH-dependent changes were not observed in the Y9F:W21F:F222W mutant. These results demonstrate that the ionization status of Tyr-9 controls the C-terminal dynamics, providing parallel pathways for product dissociation.Finally, the utility of GST-mediated hydrolysis of thiol esters formed from glutathione (GSH) and several carboxylic acid-containing drugs were exploited as a means of delivering drugs to tumors that overexpress GSTs. The competence of several human GSTs was examined, with the Pi-class GST being the most active in hydrolyzing the thiol ester formed from ethacrynic acid (EA) and GSH. Moreover, the P-class GST was inhibited and modified by the released EA. Results also indicate the importance of active site Tyr in the Alpha-, Pi-, and Mu-class GSTs. However, the exact mechanism by which this residue participates in the hydrolysis remains unknown.en_US
dc.format.extentvii, 139 p.en_US
dc.language.isoen_USen_US
dc.rightsCopyright is held by the individual authors.en_US
dc.rights.urien_US
dc.subject.otherTheses--Medicinal chemistryen_US
dc.titleThe functional role of Phe-10 and the anomalous Tyr-9 pKa in glutathione S-transferase A1-1en_US
dc.typeThesisen_US


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